1. Field of the Invention
This invention relates to a novel vaccine against Vibrio vulnificus, an opportunistic pathogen of aquaculture-raised fish and also of humans.
2. Description of the Relevant Art
Vibrio vulnificus is a Gram-negative halophilic bacterium commonly associated with estuarine and marine environments worldwide (Strom and Paranjpye. 2000. Microbes and Infection 2:177-188). This pathogen has the ability to cause severe infections in humans following the consumption of raw seafood contaminated with V. vulnificus, and thus presents a food safety issue. Wound infections can also occur through exposure of open wounds to the bacterium (Jones and Oliver. 2009. Infect. Immun. 77:1723-1733).
In addition to being an opportunistic human pathogen, V. vulnificus can cause infections in economically important aquaculture-raised fish species. V. vulnificus infections in fish are most often associated with aquaculture-reared eels Anguilla anguilla and A. japonica (Fouz et al. 2006. J. Fish Dis. 29:285-291; Tison et al. 1982. Appl. Environ. Microbiol. 44: 640-646). However, there are also reports of this pathogen causing disease in pompano Trachinotus ovatus (Li et al. 2006. Aquaculture 261-17-25) and tilapia Oreochromis spp. under production conditions (Sakata and Hattori. 1988. Fish Pathol. 23:33-40; Chen et al. 2006. J. World Aquacult. Soc. 37: 82-88). Vibrio vulnificus was re-isolated from dead rainbow trout (Oncorhynchus mykiss) reared in marine production systems; however, the role of the bacterium in causing disease was uncertain (Pederson et al. 2008. J. Fish Dis. 31:659-667). Laboratory studies have demonstrated the susceptibility of other species, such as turbot Psetta maxima, sea bass Dicentrachus labrax, and rainbow trout (Biosca and Amaro. 1996. Appl. Environ. Microbiol. 62: 2331-2337; Fouz et al. 2002. Aquacult 212:21-30). With the ever increasing importance of cultured marine and freshwater fish as a source of protein, the potential for increased disease due to Vibrio spp. is recognized (Mlandineo and Miletic. 2008. World Aquaculture Magazine 39:26-29; Mahmud et al. 2010. Appl. Environ. Microbiol. 76: 4890-4895).
V. vulnificus exhibits a large degree of phenotypic and genotypic heterogeneity. Isolates can be classified into three biotypes based on biochemical characteristics (reviewed in Jones and Oliver, supra). However, although biochemical characteristics are commonly used to assign an isolate to a particular biotype, they are variable and may not be adequate for biotyping (Sanjuán et al. 2009. Appl. Environ. Microbiol. 75: 1604-1613). For example, biotype 1 isolates are commonly associated with human infection; however, isolates from any of the three biotypes have the potential to cause disease in humans. Most isolates that cause disease in fish have been reported as biotype 2 (Biosca and Amaro, supra; Fouz et al. 2002, supra; Fouz et al. 2010. J. Fish Dis. 33:383-390).
The genetic heterogeneity observed among V. vulnificus isolates has allowed for the development of molecular methods to characterize isolates and these have been useful to discriminate between isolates with human-pathogenic potential (i.e., clinical isolates) and environmental isolates. Three such methods include restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (Aznar et al. 1994. Int. J. Systematic Bacteriol. 44:330-337; Nilsson et al. 2003. J. Clin. Microbiol. 41:442-446), polymerase chain reaction (PCR) analysis of the hemolysin gene, vvhA (Senoh et al. 2005. Microbiol. Immunol. 49: 513-519), and PCR analysis of a virulence-correlated gene, vcg (Rosche et al. 2005. Microbiol. Immunol. 49: 381-389; Warner and Oliver. 2008. Foodborne Path. Dis. 5:691-693). Recently, Sanjuán et al. (supra) evaluated these three methods for their ability to distinguish between clinical and environmental isolates. Based on the results, the authors proposed three genotypic profiles: (1) vcg type C, 16S rRNA type B, and vvhA type 1, which included biotype 1 strains from human septicemia and oyster; (2) vcg type E, 16S rRNA type A, vvhA type 2, which included biotype 2 isolates and biotype 1 isolates from fish and water and some human isolates; and (3) vcg type E, 16S rRNA type AB, vvhA type 2, which included only biotype 3 isolates. However, in addition, they also reported atypical isolates which did not fall into these three genotypic profiles and concluded from their studies that no genotyping system was able to distinguish either clinical strains from environmental strains or biogroups within the species V. vulnificus, suggesting to them that new typing methodologies useful for public health had to be developed for this particular bacterial species.
Most literature has suggested that V. vulnificus isolates pathogenic to fish, including tilapia, are biotype 2 (Biosca and Amaro, supra; Fouz et al. 2002, supra; Fouz et al. 2010, supra). Laboratory studies in five species of fish (warm and cold-water) with four biotype 1 isolates supported this because infection with these isolates did not induce mortality in fish (Biosca and Amaro, supra). However, there are a few reports of V. vulnificus isolates, exhibiting similarities to biotype 1, causing disease in finfish. Sakata and Hattori (supra) isolated strains of V. vulnificus from diseased tilapia and suggested that they were similar to biotype 1 strains based on biochemical reactions. Chen et al. (supra) characterized V. vulnificus isolates from diseased tilapia in Taiwan and suggested that the isolates were similar to biotype 1 isolates based on 16S rRNA gene sequences, but these isolates produced unique biochemical reactions compared to other human clinical isolates. Li et al. (supra) characterized an isolate of V. vulnificus obtained from diseased pompano. Their isolate exhibited unique biochemical characteristics compared to all three biotypes, but 16S rRNA gene sequencing indicated that it was closely related to a biotype 1 isolate. Since definitive genotyping was not carried out on any of the aforementioned isolates of V. vulnificus, no further information on other identifying characteristics is available.
A recent study characterized V. vulnificus strains associated with tilapia aquaculture in Bangladesh and the results indicated that all the strains were biotype 1 and similar to human clinical isolates based on genotyping (Mahmud et al., supra). The authors hypothesized that environmental factors and aquaculture practices may contribute to the emergence of more virulent isolates (Mahmud et al., supra). The possibility of V. vulnificus (biotype 1 and human clinical genotype) emerging as a fish pathogen should not be overlooked as this may present a significant disease problem for aquaculture-reared fish species and an increased risk for food safety issues related to susceptible individuals handling and consuming raw fish harboring such isolates.
In the present study, we report on the isolation of V. vulnificus from diseased hybrid tilapia (O. niloticus X O. aureus) under production in a North American water reuse aquaculture facility. Given the facts that aquaculture of marine and freshwater fish has increased and that the increased numbers of cultured fish have become an important source of protein, there is a need to address the possibility of increased disease due to Vibrio spp. and there is a need to provide vaccines for protection against Vibrio-induced diseased fish.